Cardiomyocytes derived from human-induced pluripotent stem cells (iPS-CMs) have numerous advantages for drug screening and disease modeling but present with structural and functional immaturity features. Experiments in cardiomyocytes isolated from adult human or animal hearts are conditioned by their typical rod-shaped morphology.

Here, we aimed at improving iPS-CMs architectural maturity by using a rod-shaped cell micropatterned substrate made of repeated rectangles of 120μm-length and 30μm-width and surrounded with a chemical cell-repellent.

Cardiomyocyte differentiation was performed using a chemically defined method. Thirty-five days later the generated iPS-CMs were cultured on unpatterned vs. rod-shaped micropatterned substrates for 10 additional days. We performed molecular investigations by qPCR and immunofluorescence, and electrophysiological characterization by patch-clamp technique.

The culture of micropatterned substrates resulted in the generation of rod-shaped iPS-CMs with a 4:1 cell ratio, with a more organized troponin T staining pattern exhibiting sarcomere-type striations and higher sarcomere organization score (0.25±0.07 vs. 0.13±0.004, P<0.0001). The rod-shaped iPS-CMs also showed significant improvements in electrophysiological parameters with a decrease of the spontaneous beating rate and a more hyperpolarized membrane diastolic potential (−68.11±6.25 vs. −59.17±4.94mV, P<0.001) as compared to unpatterned iPS-CMs. Rod-shaped iPS-CMs displayed a more predominant ventricular-like type after analyzing action potential recordings (91%±1), with an increase in the percentage of late ventricular-like APs compared to unpatterned (64% vs. 29%), suggesting that rod-shaped morphology stimulates ventricular maturation. Notably, the maximum upstroke velocity (dV/dtmax) (98.09±32.63 vs. 59.71±50.43V/ms, P<0.1) and the sodium current density (INa) was increased (−89.30±25.09 vs. −64.92±21.63 pA/pF, P<0.001) in rod-shaped iPS-CMs compared to unpatterned CMs. Importantly, we found no significant change in the RNA expression of the main ion channels or in major sarcomeric components. We are now assessing the Ca2+signaling properties by confocal analysis.

We developed a novel method to generate rod-shaped iPS-CMs, leading to improved functional characteristics. Preliminary results suggest that the architectural re-arrangement is the primary driver of the improved functionality observed in micropatterned iPS-CMs.